The focus of this research is the development of differentiated stem-cell based methods to repair focal cartilage damage. The long-term goal is to reliably repair joint surface injury and ultimately avoid degenerative osteoarthritis. In adults, the meager intrinsic cartilage repair following focal injury often fails to form a durable articular surface. This results from the absence of a recruitable local stem cell niche that would contribute to repair. Applying undifferentiated or poorly differentiated mesenchymal stem cells (MSC) to a joint surface in hope of achieving durable repair has been ineffective;controlled studies in large animal models show cultured MSCs have only transitory impact on cartilage repair. This project will develop a reliable mechanism to affect a chromosomal based shift in stem cell phenotypic programming that may drive in vivo formation of a dedicated stem cell niche in cartilage. This proposal will investigate the effect of autologous MSCs stably transduced with chondroinductive genes from the Sox transcription factor and TGF- b superfamilies to derive a cartilage progenitor stem cell niche. The hypothesis is that creating a "pre- programmed" stem cell niche in cartilage repair tissue will enhance chondrogenic repopulation and organized repair in acute cartilage injury. Equine Sox transcription factors Sox5, Sox6 and Sox9, the master controllers of chondrogenesis, will be compared separately and in combination. Genes for each equine Transcription Factor (TF) will be inserted into Sleeping Beauty (SB) transposable elements, and assessed by monolayer selection of integrants, and a transwell 3D culture system, to determine chondrogenic index through gene expression profiling, immunohistochemistry, and biochemical assays. The TGF superfamily members TGF-b1, -b2, and -b3 will then be compared by development of similar SB constructs. A combination of the best of the Sox TF and TGF-p gene transduction outcomes will then be evaluated together in culture and then in vivo to allow a critical analysis of long term final cartilage phenotype. Persistence of the Sox and TGF-p, expression and subsequent cartilage marker gene profiling will be determined by qPCR, and translation of cartilage matrix gene message confirmed by assay of aggrecan and collagen type II. PUBLIC HEALTH RELEVANCE: Joint injuries are a frustrating aspect of athletics and aging. Conservative estimates of the annual financial burden of cartilage injury and subsequent arthritis in the US alone approach 20 billion dollars annually. Mechanisms that consistently induce the production of normal cartilage within injured sites will provide significant improvement in joint repair and arthritis prevention.